U.S. patent application number 15/509014 was filed with the patent office on 2017-09-28 for composition comprising (6s)-5-methyl tetrahydrofolic acid or salt thereof, and preparation and use thereof.
This patent application is currently assigned to LIANYUNGANG JINKANG HEXIN PHARMACEUTICAL CO., LTD.. The applicant listed for this patent is LIANYUNGANG JINKANG HEXIN PHARMACEUTICAL CO., LTD.. Invention is credited to Yongzhi CHENG, Zhi CHENG.
Application Number | 20170273984 15/509014 |
Document ID | / |
Family ID | 55439151 |
Filed Date | 2017-09-28 |
United States Patent
Application |
20170273984 |
Kind Code |
A1 |
CHENG; Yongzhi ; et
al. |
September 28, 2017 |
COMPOSITION COMPRISING (6S)-5-METHYL TETRAHYDROFOLIC ACID OR SALT
THEREOF, AND PREPARATION AND USE THEREOF
Abstract
Disclosed are a composition including (6S)-5-methyl
tetrahydrofolic acid or a salt thereof, and preparation and use
thereof. In the composition, the content of (6S)-5-methyl
tetrahydrofolic acid or the salt thereof is not less than 98.0%,
the content of a related impurity JK12A is not greater than 0.1%,
and 5-methyl tetrahydropterioic acid is not detectable.
Inventors: |
CHENG; Yongzhi;
(Lianyungang, CN) ; CHENG; Zhi; (Lianyungang,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIANYUNGANG JINKANG HEXIN PHARMACEUTICAL CO., LTD. |
Lianyungang, Jiangsu |
|
CN |
|
|
Assignee: |
LIANYUNGANG JINKANG HEXIN
PHARMACEUTICAL CO., LTD.
Lianyungang, Jiangsu
CN
|
Family ID: |
55439151 |
Appl. No.: |
15/509014 |
Filed: |
September 4, 2015 |
PCT Filed: |
September 4, 2015 |
PCT NO: |
PCT/CN2015/088925 |
371 Date: |
March 6, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/519 20130101;
A61P 3/02 20180101; A61P 25/00 20180101; A61P 27/02 20180101; A61P
25/24 20180101; A61P 37/06 20180101; A61P 7/06 20180101; A61K 47/22
20130101; C07D 475/04 20130101; A61P 19/02 20180101; A61P 15/08
20180101; A61P 9/10 20180101; A61P 9/00 20180101; A61P 21/00
20180101; A61P 9/12 20180101; A61K 9/08 20130101; A61P 43/00
20180101; A61P 25/28 20180101; A61P 1/00 20180101; A61P 29/00
20180101; A61P 17/06 20180101; A61P 37/02 20180101; A61P 39/02
20180101; A61P 15/06 20180101 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A61K 47/22 20060101 A61K047/22 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 4, 2014 |
CN |
201410449916.5 |
Claims
1. A composition comprising (6S)-5-methyl tetrahydrofolic acid or a
salt thereof, wherein the content of (6S)-5-methyl tetrahydrofolic
acid or the salt thereof in the composition is not less than 98.0%,
the content of a related impurity JK12A is not greater than 0.1%,
and 5-methyl tetrahydropterioic acid is not detectable
##STR00007##
2. The composition comprising (6S)-5-methyl tetrahydrofolic acid or
a salt thereof according to claim 1, wherein the content of
(6S)-5-methyl tetrahydrofolic acid or the salt thereof is not less
than 99.0%, the content of the related impurity JK12A is not
greater than 0.1%, and 5-methyl tetrahydropterioic acid is not
detectable.
3. The composition comprising (6S)-5-methyl tetrahydrofolic acid or
a salt thereof according to claim 1, wherein the salt of
(6S)-5-methyl tetrahydrofolic acid is selected from a potassium
salt, a sodium salt, a calcium salt, a magnesium salt, a barium
salt, a zinc salt, a D-glucosamine salt, a D-galactosamine salt, or
an arginine salt.
4. A method for preparing a composition comprising high-purity
(6S)-5-methyl tetrahydrofolic acid or a salt thereof according to
claim 1, the method comprising a method A or a method B, wherein
the method A comprises reacting a crude salt of (6S)-5-methyl
tetrahydrofolic acid with a reverse reducing agent in water at pH
6-8 at a temperature of 50-90.degree. C. directly, or reacting
ultrasonically, to obtain a composition comprising the salt of
(6S)-5-methyl tetrahydrofolic acid after reaction; and the method B
comprises reacting crude (6S)-5-methyl tetrahydrofolic acid, a
reverse reducing agent, and a corresponding salt in water at pH 6-8
at a temperature of 50-90.degree. C., or reacting ultrasonically,
to obtain a composition comprising a salt of (6S)-5-methyl
tetrahydrofolic acid after reaction; wherein the reverse reducing
agent is one or more selected from vitamin C, an ester of vitamin
C, a salt of vitamin C, isovitamin C, an ester of isovitamin C, a
salt of isovitamin C, mercaptoethanol, cysteine, mercaptoethane
sulfonic acid, dithiothreitol, reductive glutathione, or zinc
sulfate.
5. The method for preparing a composition comprising high-purity
(6S)-5-methyl tetrahydrofolic acid or a salt thereof according to
claim 4, wherein the reverse reducing agent is one or more selected
from vitamin C, an ester of vitamin C, a salt of vitamin C,
isovitamin C, an ester of isovitamin C, or a salt of isovitamin C
and the corresponding salt is a chloride.
6. The method for preparing a composition comprising high-purity
(6S)-5-methyl tetrahydrofolic acid or a salt thereof according to
claim 4, wherein during the reaction of the method A or B, the
reaction temperature is 60-80.degree. C., and the pH is 7-7.5 and
adjusted with sodium hydroxide; and the concentration of the
reverse reducing agent in water is not less than 2%, by weight.
7. A solid solution comprising (6S)-5-methyl tetrahydrofolic acid
or a salt thereof and a stabilizing agent, wherein the solid
solution is composed essentially of (6S)-5-methyl tetrahydrofolic
acid or a salt thereof and the stabilizing agent, and the total
content of the stabilizing agent and (6S)-5-methyl tetrahydrofolic
acid or the salt thereof in a liquid chromatogram of the solid
solution is greater than 99.5%, wherein the stabilizing agent is
one or more selected from vitamin C, an ester of vitamin C, a salt
of vitamin C, isovitamin C, an ester of isovitamin C, a salt of
isovitamin C, dithiothreitol, reductive glutathione, or zinc
sulfate.
8. The solid solution comprising (6S)-5-methyl tetrahydrofolic acid
or a salt thereof and a stabilizing agent according to claim 7,
wherein the total content of the stabilizing agent and
(6S)-5-methyl tetrahydrofolic acid or the salt thereof in a liquid
chromatogram of the solid solution is greater than 99.8%.
9. The solid solution comprising (6S)-5-methyl tetrahydrofolic acid
or a salt thereof and a stabilizing agent according to claim 7,
wherein the stabilizing agent is one or more selected from vitamin
C, an ester of vitamin C, a salt of vitamin C, isovitamin C, an
ester of isovitamin C, or a salt of isovitamin C.
10. A method for preparing the solid solution comprising
(6S)-5-methyl tetrahydrofolic acid or a salt thereof and a
stabilizing agent according to claim 7, the method comprising a
method C or a method D, wherein the method C comprises reacting
crude (6S)-5-methyl tetrahydrofolic acid or a high-purity
(6S)-5-methyl tetrahydrofolic acid with a stabilizing agent in
water at pH 3-5 at a temperature of 50-90.degree. C. directly, or
reacting ultrasonically, and separating the solid that is
crystallized out after reaction; and the method D comprises
reacting crude (6S)-5-methyl tetrahydrofolic acid of or a
high-purity (6S)-5-methyl tetrahydrofolic acid a stabilizing agent
and a corresponding salt in water at pH 6-8 at a temperature of
50-90.degree. C., or reacting ultrasonically, and separating the
solid that is crystallized out after reaction; wherein the
stabilizing agent is one or more selected from vitamin C, an ester
of vitamin C, a salt of vitamin C, isovitamin C, an ester of
isovitamin C, a salt of isovitamin C, dithiothreitol, reductive
glutathione or zinc sulfate; and the corresponding salt is a
chloride and wherein the high-purity (6S)-5-methyl tetrahydrofolic
acid is a composition comprising (6S)-5-methyl tetrahydrofolic acid
or a salt thereof, wherein the content of (6S)-5-methyl
tetrahydrofolic acid or the salt thereof in the composition is not
less than 98.0%, the content of a related impurity JK12A is not
greater than 0.1%, and 5-methyl tetrahydropterioic acid is not
detectable ##STR00008##
11. The method for preparing a solid solution comprising
(6S)-5-methyl tetrahydrofolic acid or a salt thereof and a
stabilizing agent according to claim 10, wherein during the
reaction of the method C, the reaction temperature is 60-80.degree.
C., and the pH is 3-4 and adjusted with hydrochloric acid; and
during the reaction of the method D, the reaction temperature is
60-80.degree. C., and the pH is 7-7.5 and adjusted with sodium
hydroxide.
12. A pharmaceutical composition, comprising a) the composition
comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof
according to claim 1, or a solid solution comprising (6S)-5-methyl
tetrahydrofolic acid or a salt thereof and a stabilizing agent,
wherein the solid solution is composed essentially of (6S)-5-methyl
tetrahydrofolic acid or a salt thereof and the stabilizing agent,
and the total content of the stabilizing agent and (6S)-5-methyl
tetrahydrofolic acid or the salt thereof in a liquid chromatogram
of the solid solution is greater than 99.5%, Wherein the
stabilizing agent is one or more selected from vitamin C, an ester
of vitamin C, a salt of vitamin C, isovitamin C, an ester
isovitamin C, a salt of isovitamin C, dithiothreitol, reductive
glutathione, or zinc sulfate; and b) a reducing agent, which is one
or more selected from vitamin C, an ester of vitamin C, a salt of
vitamin C, isovitamin C, an ester of isovitamin C, a salt of
isovitamin C, mercaptoethanol, cysteine, mercaptoethane sulfonic
acid, dithiothreitol, reductive glutathione, or zinc sulfate.
13. The pharmaceutical composition according to claim 12, which is
in the form of common tablets, sustained release tablets,
controlled release tablets, effervescent tablets, granules, common
capsules, sustained release capsules, controlled release capsules,
dry suspensions, oral solutions, oral suspensions, injections, or
freeze-dried powder injections, wherein the weight ratio of the
reducing agent to the (6S)-5-methyl tetrahydrofolic acid or the
salt thereof is 0.02-1000:1.
14. A method of treating or preventing diseases associated with
deficiency of 5-methyl tetrahydrofolic acid or a salt thereof in
mammals, comprising administering to a patient in need thereof a
drug comprising (i) the composition comprising (6S)-5-methyl
tetrahydrofolic acid or a salt thereof according to claim 1, or
(ii) a solid solution comprising (6S)-5-methyl tetrahydrofolic acid
or a salt thereof and a stabilizing agent, wherein the solid
solution is composed essentially of (6S)-5-methyl tetrahydrofolic
acid or a salt thereof and the stabilizing agent, and the total
content of the stabilizing agent and (6S)-5-methyl tetrahydrofolic
acid or the salt thereof in a liquid chromatogram of the solid
solution is greater than 99.5%, wherein the stabilizing agent is
one or more selected from vitamin C, an ester of vitamin C, a salt
of vitamin C, isovitamin C, an ester of isovitamin C, a salt of
isovitamin C, dithiothreitol, reductive glutathione, or zinc
sulfate.
Description
BACKGROUND
[0001] Technical Field
[0002] The present invention relates to the technical field of
pharmaceutical preparations, and particularly to a composition
comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof, a
preparation method therefor, a solid solution composition
comprising high-purity (6S)-5-methyl tetrahydrofolic acid or a salt
thereof, a preparation method therefor, and use of (6S)-5-methyl
tetrahydrofolic acid and salts thereof, including the use of the
composition or the solid solution comprising (6S)-5-methyl
tetrahydrofolic acid or a salt thereof, in which a pharmaceutically
acceptable reductive substance is added as a stabilizing agent. The
safe method of using provides excellent safety properties of
(6S)-5-methyl tetrahydrodrofolic acid products.
[0003] Related Art
[0004] In recent years, more and more research is conducted on the
physiological activity for human of 5-methyl tetrahydrofolic acid
and salts thereof. Good application prospects are exhibited when
5-methyl tetrahydrofolic acid or a salt thereof is used as a folic
acid supplement to human for preventing and/or treating
neurological disorders; used in combination with 5-fluorouracil or
methotrexate for tumor therapy; used in the treatment of
pathophysiology of vascular and cardiovascular diseases; and used
in the therapy and adjuvant therapy of severe depression and severe
senile dementia.
[0005] However, 5-methyl tetrahydrofolic acid or a salt thereof is
quite labile, very susceptible to degradation, and highly sensitive
particularly to oxygen and moisture. Therefore, a composition
comprising 5-methyltetrahydrofolate or a salt thereof, such as a
pharmaceutical preparation, generally suffers from the problems of
poor stability, easy discoloration, darkened color, remarkably
lowered content, and increased degradation product.
[0006] Many papers concern the oxidative degradation of 5-methyl
tetrahydrofolic acid or its single active isomer (6S)-5-methyl
tetrahydrofolic acid. Unfortunately, according to a study by the
present inventors, many previous discussions on the primary
oxidation products of 5-methyl tetrahydrofolic acid are either
unilateral or even erroneous. These conclusions mislead people to
reduce the quality requirements for 5-methyl tetrahydrofolic acid.
Based on the discussions in Ratanasthien K et al. Serum folates in
man (Ratanasthien K., Blair J. A., Leeming R. J., Cooke W. T. and
Melikian V. (1977). J. Clin. Path. 30: 438-448); Blair J. A et al.
Autoxidation of 5-Methyl-5,6,7,8-tetrahydrofolic acid (Blair J. A.,
Pearson A. J. and Robb A. J. (1975). J. C. S. Perkin II, p.
18-21.); and European Food Safety Authority, Opinion of the
Scientific Panel on Food Additives, Flavourings, Processing Aids
and Materials in Contact with Food on a request from the Commission
related to Calcium L-Methylfolate Question (The EFSA Journal (2004)
135. 1-20), 5-methyl tetrahydrofolic acid is initially oxidized
into 5-methyl dihydrofolic acid in the presence of an oxidant.
##STR00001##
[0007] In other literatures, the primary oxidation product is
characterized as 4-hydroxy-5-methyl tetrahydrofolic acid. This
product is inferred to be not toxic according to the structure.
[0008] An efficient method is developed by the present inventors,
in which a high-purity primary oxidation product of 5-methyl
tetrahydrofolic acid is prepared firstly. A series of studies are
conducted (PCT/CN2013/073959) and it is found unexpectedly that the
primary oxidation product of 5-methyl tetrahydrofolic acid or a
salt thereof is JK12A having a structural formula of
##STR00002##
[0009] JK12A is a primary oxidation product of 5-methyl
tetrahydrofolic acid, which, depending on specific conditions, has
a relative retention time from the main peak of about 0.36 under
the detection conditions of the present invention, and is also
known as 5-methyl-dihydrofolic acid or
4-hydroxy-5-methyl-tetrahydrofolic acid in relevant
literatures.
[0010] It is a product of new structure, and this finding overturns
the previous conclusions, including those in the above-mentioned
literatures. In general, large changes in structure will lead to a
significant change in physiological activity. Subsequent studies
show that JK12A has a significant inhibitory effect on T lymphocyte
proliferation. It is generally believed that this causes the
reduction of human immunity.
TABLE-US-00001 T cell T proliferation lymphocyte Comprehensive Cell
Stimulus evaluation Concentration Mean Cell control with Con A
Percent determined OD SD survival Stimulus Mean CPM SD enhancement/
Control M 0.122 0.003 rate control 43977 8000 inhibition JK12A
100.000 0.089 0.002 73% 40.000 0.109 0.006 89% 43537 4110 -1% 16.0
0.113 0.007 92% 36061 241 -18% 6.4 0.103 0.007 84% 30784 3903 -30%
2.56 0.076 0.008 62% 25507 10004 -42% 1.024 0.073 0.014 59% 17590
8005 -60% 0.41 0.052 0.015 75% 14073 10299 -68% 0.164 40459 7501
-8%
[0011] An acute toxicity study of JK12A shows that at a dose of
2000 mg/kg, the test mice are killed on the spot within 1 minute.
However, the anatomical findings show no significant changes in
both the liver and the kidney, suggesting that the compound may
possibly toxically target other tissues or organs and has a
significant acute toxicity.
[0012] The present inventors find through studies that 5-methyl
tetrahydropterioic acid is another common impurity in the product
5-methy tetrahydrofolic acid. Similar to the formation of JK12A,
the impurity is oxidized to form JK1303 having a structure
below
##STR00003##
[0013] Subsequent studies have found that JK1303 has serious renal
toxicity. 14 days after the mice are given a single dose of JK1303
at a dosage of 1000 mg/kg, severe renal tubular necrosis of the
mice occurs, and a large number of vacuoles are anatomically found
in the renal tubules. The toxicity of the primary oxidation product
of 5-methyl tetrahydropterioic acid is also significantly
changed.
[0014] The above experiments show that in a product containing
5-methyl tetrahydrofolic acid and its salt, the impurity JK12A
produced from the oxidative degradation of 5-methyl tetrahydrofolic
acid and its salt, and the oxidation product JK1303 of 5-methyl
tetrahydropterioic acid are very harmful to human body, and this
cannot and should not be ignored. Therefore, it is necessary to
develop a solution for using 5-methyl tetrahydrofolic acid or its
salt safely and reliably.
[0015] Chinese Patent No. CN102612358 discloses particles of
stabilized salt of 5-methyl tetrahydrofolic acid, in which a
crystalline salt of 5-methyl tetrahydrofolic acid is embedded in a
wax to increase the stability.
[0016] Chinese Patent No. CN102813656 discloses a composition
comprising stabilized salt of 5-methyl tetrahydrofolic acid, in
which one or more amino acids free of mercapto groups are contained
to increase the stability.
[0017] U.S. Pat. No. 6,441,168 discloses that the stability of
5-methyl tetrahydrofolic acid calcium salt is considerably
increased by preparing it into a crystal form. However, the crystal
form has a large particle size, is dissolved slowly, and needs to
be ground first during the production of preparations, which trends
to increase the risk of oxidation of 5-methyl tetrahydrofolic
acid.
[0018] Chinese Patent No. CN2012086794 discloses a new crystal form
of (6S)-5-methyl tetrahydrofolic acid calcium salt, which has good
chemical stability and greatly increased solubility, such that no
grinding is needed during the production of preparations. However,
the content of the impurities such as JK12A is still high, and
trends to increase gradually with the elapse of time.
[0019] It is also reported in literatures that 5-methyl
tetrahydrofolic acid is embedded in microcapsules prepared with
cellulose, shellac and other materials, to improve the stability of
the active ingredients 5-methyl tetrahydrofolic acid in the
preparation product.
[0020] However, the content of JK12A still cannot be controlled at
a level that is safe enough by all these measures. For the raw
medicine products of 5-methyl tetrahydrofolic acid commercially
available at present, the total content of the impurities in a
crystal form of 5-methyl tetrahydrofolic acid is generally in the
range of 1-4%, in which the content of JK12A is up to 0.3-3.0%. In
commercially available amorphous products, the total content of the
impurities is as high as 10% or above, causing a serious threat to
the safety of drugs during use. These facts show that the
impurities including JK12A contained in all of these raw medicine
products are not properly treated at present. The high chemical
lability also makes it difficult to precisely control the dosage
during use. For example, at a common dose of 0.451 mg, lability
brings about a large deviation to the actual dose.
SUMMARY
[0021] In view of the prior art, an objective of the present
invention is to provide a composition comprising high-purity
(6S)-5-methyl tetrahydrofolic acid or a salt thereof, and a
preparation method therefor.
[0022] Another objective of the present invention is to provide a
solid solution comprising (6S)-5-methyl tetrahydrofolic acid or a
salt thereof and a stabilizing agent, and a preparation method
therefor.
[0023] A third objective of the present invention is to provide a
pharmaceutical composition comprising high-purity (6S)-5-methyl
tetrahydrofolic acid or a salt thereof, or the solid solution.
[0024] A fourth objective of the present invention is to provide
use of the high-purity (6S)-5-methyl tetrahydrofolic acid or a salt
thereof, or the solid solution comprising (6S)-5-methyl
tetrahydrofolic acid or a salt thereof and a stabilizing agent, in
medical areas.
[0025] The objectives of the present invention can be accomplished
through the following.
[0026] A composition comprising (6S)-5-methyl tetrahydrofolic acid
or a salt thereof is provided, in which the content of
(6S)-5-methyl tetrahydrofolic acid or the salt thereof is not less
than 98.0%, the content of a related impurity JK12A is not greater
than 0.1%, and 5-methyl tetrahydropterioic acid is not
detectable.
##STR00004##
[0027] The composition comprising (6S)-5-methyl tetrahydrofolic
acid or a salt thereof according to the present invention is
composed essentially of (6S)-5-methyl tetrahydrofolic acid or a
salt thereof. The content of (6S)-5-methyl tetrahydrofolic acid or
the salt thereof in the composition is a content in percentages by
weight of (6S)-5-methyl tetrahydrofolic acid or the salt thereof,
or a content as detected by HPLC, and generally a content in
weight.
[0028] In an embodiment, the content of (6S)-5-methyl
tetrahydrofolic acid or the salt thereof in the composition is not
less than 99.0%, the content of the related impurity JK12A is not
greater than 0.1%, and 5-methyl tetrahydropterioic acid is not
detectable.
[0029] In another embodiment, the content of (6S)-5-methyl
tetrahydrofolic acid or the salt thereof in the composition is not
less than 99.5%, the content of the related impurity JK12A is not
greater than 0.1%, and 5-methyl tetrahydropterioic acid is not
detectable.
[0030] In another embodiment, the content of (6S)-5-methyl
tetrahydrofolic acid or the salt thereof in the composition is not
less than 99.8%, the content of the related impurity JK12A is not
greater than 0.05%, and 5-methyl tetrahydropterioic acid is not
detectable.
[0031] The salt of (6S)-5-methyl tetrahydrofolic acid in the
present invention is selected from a potassium salt, a sodium salt,
a calcium salt, a magnesium salt, a barium salt, a zinc salt, a
D-glucosamine salt, a D-galactosamine salt, or an arginine salt,
and preferably a calcium salt, a D-glucosamine salt, a
D-galactosamine salt or an arginine salt.
[0032] A method for preparing the composition comprising
(6S)-5-methyl tetrahydrofolic acid or a salt thereof is provided.
The method includes a method A or a method B.
[0033] The method A includes reacting a crude salt of (6S)-5-methyl
tetrahydrofolic acid with a reverse reducing agent in water at pH
6-8 at a temperature of 50-90.degree. C. directly, or reacting
ultrasonically, to obtain a composition comprising a salt of
(6S)-5-methyl tetrahydrofolic acid after reaction;
[0034] The method B includes reacting crude (6S)-5-methyl
tetrahydrofolic acid, a reverse reducing agent, and a corresponding
salt in water at pH 6-8 at a temperature of 50-90.degree. C., or
reacting ultrasonically, to obtain a composition comprising a salt
of (6S)-5-methyl tetrahydrofolic acid after reaction
[0035] In the method A or B, the reverse reducing agent is one or
more selected from vitamin C, an ester of vitamin C, a salt of
vitamin C, isovitamin C, an ester of isovitamin C, a salt of
isovitamin C, mercaptoethanol, cysteine, mercaptoethane sulfonic
acid, dithiothreitol, reductive glutathione, or zinc sulfate.
[0036] In a preferred embodiment, the reverse reducing agent in the
method A or B is one or more selected from vitamin C, an ester of
vitamin C, a salt of vitamin C, isovitamin C, an ester of
isovitamin C, or a salt of isovitamin C, and preferably one or more
of vitamin C, a sodium salt of vitamin C, a potassium salt of
vitamin C, a calcium salt of vitamin C, a magnesium salt of vitamin
C phosphate, a sodium salt of vitamin C phosphate, or isovitamin C,
and most preferably vitamin C or isovitamin C; and the
corresponding salt is a chloride, and preferably calcium chloride,
magnesium chloride, and zinc chloride.
[0037] In a preferred embodiment, during the reaction of the method
A or B, the reaction temperature is 60-80.degree. C., and the pH is
7-7.5 and adjusted preferably with sodium hydroxide; and the
concentration of the reverse reducing agent in water is not less
than 2%, preferably not less than 20%, and further preferably not
less than 40%, by weight.
[0038] A solid solution comprising (6S)-5-methyl tetrahydrofolic
acid or a salt thereof and a stabilizing agent is provided, where
the solid solution is composed essentially of (6S)-5-methyl
tetrahydrofolic acid or a salt thereof and the stabilizing agent.
The total content of the stabilizing agent and (6S)-5-methyl
tetrahydrofolic acid or the salt thereof in a liquid chromatogram
of the solid solution is greater than 99.5%. The stabilizing agent
is one or more selected from vitamin C, an ester of vitamin C, a
salt of vitamin C, isovitamin C, an ester of isovitamin C, a salt
of isovitamin C, dithiothreitol, reductive glutathione, or zinc
sulfate.
[0039] In a preferred embodiment, the total content of the
stabilizing agent and (6S)-5-methyl tetrahydrofolic acid or the
salt thereof in a liquid chromatogram of the solid solution
comprising (6S)-5-methyl tetrahydrofolic acid or the salt thereof
and the stabilizing agent is greater than 99.8%, and preferably
greater than 99.9%. The (6S)-5-methyl tetrahydrofolic acid or the
salt thereof is the high-purity (6S)-5-methyl tetrahydrofolic acid
or a salt thereof according to claim 1.
[0040] In a preferred embodiment, the stabilizing agent is one or
more selected from vitamin C, an ester of vitamin C, a salt of
vitamin C, isovitamin C, an ester of isovitamin C, or a salt of
isovitamin C, preferably one or more of vitamin C, a sodium salt of
vitamin C, a potassium salt of vitamin C, a calcium salt of vitamin
C, a magnesium salt of vitamin C phosphate, a sodium salt of
vitamin C, isovitamin C, a sodium salt of isovitamin C, a potassium
salt of isovitamin C, a calcium salt of isovitamin C, a magnesium
salt of isovitamin C phosphate, or a sodium salt of isovitamin C
phosphate, and further preferably vitamin C, or isovitamin C. The
weight ratio of the stabilizing agent to (6S)-5-methyl
tetrahydrofolic acid or the salt thereof is 0.2-1000:1, and
preferably 1-10:1.
[0041] A method for preparing the solid solution comprising
(6S)-5-methyl tetrahydrofolic acid or a salt thereof and a
stabilizing agent is provided. The method includes a method C or a
method D.
[0042] The method C includes: reacting crude (6S)-5-methyl
tetrahydrofolic acid or the high-purity (6S)-5-methyl
tetrahydrofolic acid according to claim 1 with a stabilizing agent
in water at pH 3-5 at a temperature of 50-90.degree. C. directly,
or reacting ultrasonically, and separating the solid that is
crystallized out after reaction.
[0043] The method D includes: reacting crude (6S)-5-methyl
tetrahydrofolic acid or the high-purity (6S)-5-methyl
tetrahydrofolic acid according to claim 1, a stabilizing agent and
a corresponding salt in water at pH 6-8 at a temperature of
50-90.degree. C., or reacting ultrasonically, and separating the
solid that is crystallized out after reaction.
[0044] The stabilizing agent is one or more selected from vitamin
C, an ester of vitamin C, a salt of vitamin C, isovitamin C, an
ester of isovitamin C, a salt of isovitamin C, dithiothreitol,
reductive glutathione or zinc sulfate; and the corresponding salt
is a chloride, and preferably calcium chloride, magnesium chloride,
and zinc chloride
[0045] In a preferred embodiment, during the reaction of the method
C, the reaction temperature is 60-80.degree. C., and the pH is 3-4
and adjusted with hydrochloric acid; and during the reaction of the
method D, the reaction temperature is 60-80.degree. C., and the pH
is 7-7.5 and adjusted with sodium hydroxide
[0046] A pharmaceutical composition is provided, which
comprises:
[0047] a) the composition comprising (6S)-5-methyl tetrahydrofolic
acid or a salt thereof, or the solid solution comprising
(6S)-5-methyl tetrahydrofolic acid or a salt thereof and a
stabilizing agent; and
[0048] b) a reducing agent, which is one or more selected from
vitamin C, an ester of vitamin C, a salt of vitamin C, isovitamin
C, an ester of isovitamin C, a salt of isovitamin C,
mercaptoethanol, cysteine, mercaptoethane sulfonic acid,
dithiothreitol, reductive glutathione, or zinc sulfate.
[0049] In an embodiment, the pharmaceutical composition is in the
form of common tablets, sustained release tablets, controlled
release tablets, effervescent tablets, granules, common capsules,
sustained release capsules, controlled release capsules, dry
suspensions, oral solutions, oral suspensions, injections, or
freeze-dried powder injections.
[0050] In an embodiment, the weight ratio of the reducing agent to
the (6S)-5-methyl tetrahydrofolic acid or the salt thereof in the
pharmaceutical composition is 0.2-1000:1, and preferably
1-10:1.
[0051] Use of the high-purity (6S)-5-methyl tetrahydrofolic acid or
a salt thereof, or the solid solution comprising (6S)-5-methyl
tetrahydrofolic acid or a salt thereof and a stabilizing agent
according to the present invention in the preparation of drugs for
treating or preventing diseases associated with deficiency of
5-methyl tetrahydrofolic acid or a salt thereof in mammals.
DEFINITION OF TERMS
[0052] The terms used in the present invention have the following
meanings, unless particularly specified otherwise.
[0053] Reversion refers to a phenomenon in which the impurity JK12A
in (6S)-5-methyl tetrahydrofolic acid or a salt thereof is
reversely converted into (6S)-5-methyl tetrahydrofolic acid or a
salt thereof; and JK1303 is reversely converted into 5-methyl
tetrahydropterioic acid.
[0054] Salting out is a means of improving the crystallization by
adding a certain concentration of neutral salt to a solution
containing a solid solute, thereby increasing the yield and
quality.
[0055] Solid solution refers to a mixture formed by dispersing one
or more solids uniformly in another solid.
[0056] Related substances refer to impurities introduced and
produced during the production, use, and storage of (6S)-5-methyl
tetrahydrofolic acid and salts thereof, including
4-aminobenzoylglutamic acid, JK12A, (6S)-Mefox, pyrazino-triazine
derivatives, tetrahydrofolic acid, 7,8-dihydrofolic acid, folic
acid, 5,10-methyl tetrahydrofolic acid, 5-methyl tetrahydropterioic
acid, N2-methylamino-tetrahydrofolic acid, and salts thereof.
[0057] Purity refers to the proportion of the peak area of
(6S)-5-methyl tetrahydrofolic acid to the total area of
(6S)-5-methyl tetrahydrofolic acid and all the related substances
in the liquid chromatogram.
[0058] Content as used herein refers to percentages by weight of a
material, unless particularly specified otherwise.
[0059] In previous studies, attempts are made to remove the oxygen
by adding some oxygen scavengers such as mercaptoethanol, ascorbic
acid, sodium sulfite and so on, to the solution
(6S)-5-methyltetrahydrofolic acid, thereby preventing sustained
oxidative degradation. However, the use of these substances in
production is unfeasible and unnecessary. In these studies, the
purpose of oxygen removal can be achieved by the use of low
concentrations of reducing agent. However, sufficient reversion of
the oxidized impurities as described in this patent cannot be
achieved with such a concentration. This is modified by the use of
high concentrations of reducing agents in the METHOD JK.
[0060] The present inventors surprisingly find that the use of a
high concentration of a reducing agent with moderate reducibility
can achieve the reversion of most of the impurities produced from
oxidative degradation, such as reversion of JK12A to (6S)-5-methyl
tetrahydrofolic acid, and reversion of JK1303 to 5-methyl
tetrahydropterioic acid. High-purity (6S)-5-methyl tetrahydrofolic
acid or a salt thereof is crystallized out by means of salting out
that follows. In this way, an incredibly high purity is obtained
with a high yield, which has not been reported in the
literature.
##STR00005##
[0061] (6S)-5-methyl tetrahydrofolic acid or a salt thereof having
a purity of 98.0% or higher, or even approximate 100% can be
prepared through this method. The content of related impurities
including JK12A, JK1303, and 5-methyl tetrahydropterioic acid is
greatly reduced even to a level below the detection limit of liquid
chromatography, which is impossible to be achieved in the prior
art.
[0062] The significance of the successful preparation of
high-purity (6S)-5-methyl tetrahydrofolic acid and salts thereof
lies not only in the significant progress in the manufacturing
technology, but also in the correcting and avoiding of the
consequences resulting from the previous misunderstanding with
respect to the quality requirement for (6S)-5-methyl
tetrahydrofolic acid and salts thereof due to the lack of research.
With the gradual increase in the dosage of (6S)-5-methyl
tetrahydrofolic acid, this will be more obvious. The concern in
this regard can be explained by the fact that as for the acute
toxicity test data published at present, the LD50 (median lethal
dose) of calcium (6S)-5-methyltetrahydrofolate is 2000 mg/kg; and
after the harmful impurities in the product are eliminated by using
the technology of the present invention, the mice have no abnormal
response at a dosage of 15000 mg/kg in a comparative test, so it is
concluded that the maximum tolerable dose (MTh) is greater than 15
g/kg. Therefore, the contribution of the new technology to the
product safety is self-evident
[0063] The present invention also provides a method for preparing a
composition of (6S)-5-methyl tetrahydrofolic acid or a salt
thereof. The method comprises dissolving crude (6S)-5-methyl
tetrahydrofolic acid or a salt thereof in water or an aqueous
solvent, treating with a reverse reducing agent, to reverse most of
the impurities produced from oxidative degradation into
(6S)-5-methyl tetrahydrofolic acid or a salt thereof, and then
crystallizing high-purity (6S)-5-methyl tetrahydrofolic acid or a
salt thereof out by means of salting out or other process.
[0064] The reverse reducing agent useful in the present invention
is as described above, and the reverse reducing agent for reversing
the oxidized impurities needs to have suitable reducibility. If the
reducibility is too high, reductive degradation is caused; and if
the reducibility is too low, no desirable effect can be
achieved.
[0065] In an embodiment, in the methods A and B and in the
preparation of the composition above, the content of the reverse
reducing agent in the solvent is not less than 2%, preferably not
less than 20%, and further preferably not less than 40%, by
weight.
[0066] The term salting out as used above has a meaning that is
slightly different from that generally understood in biochemistry,
but the effect is similar, in that the yield of crystals is
increased, and the enrichment effect from the mother liquor for
crystallization against the impurity is enhanced, so that the
purity of the product reaches an incredible level.
[0067] The salting-out agent serving for salting out used in the
present invention includes, in addition to the reducing agent
above, a commonly used salt, for example neutral inorganic salts
such as sodium chloride, potassium chloride, and magnesium sulfate.
In the present invention, the salting-out effect is preferably
achieved by increasing the concentration of the reducing agent.
[0068] Moreover, in a liquid chromatogram of the solid solution
comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof and
a stabilizing agent provided in the present invention, total
content of the stabilizing agent and (6S)-5-methyl tetrahydrofolic
acid or the salt thereof is greater than 99.5%, preferably greater
than 99.8%, and further preferably greater than 99.9%. The content
is calculated based on the peak area in the liquid
chromatogram.
[0069] The solid solution has an excellent homogeneous dispersion,
and can effectively protect the active ingredient of the present
invention.
[0070] In the solid solution of the present invention, the
(6S)-5-methyl tetrahydrofolic acid and the stabilizing agent are
kept highly homogeneous, thus ensuring the stabilization effect. In
a typical embodiment, the (6S)-5-methyl tetrahydrofolic acid
calcium salt is of crystal form C with uniform and fine particles
and narrow particle size distribution, and can form a highly
homogeneous solid solution with the stabilizing agent. This is
difficult to be achieved in the prior art.
[0071] In a preferred embodiment, the solid solution of the present
invention is a solid solution of (6S)-5-methyl tetrahydrofolic acid
or a salt with vitamin C, isovitamin C, zinc sulfate, reductive
glutathione, dithiothreitol or a salt thereof.
[0072] In a typical case, the solid solution prepared according to
the present invention has only two typical absorption peaks in the
HPLC chromatogram, and is thus highly neat.
[0073] The stabilizing agent and the (6S)-5-methyl tetrahydrofolic
acid are homogeneously dispersed in the solid solution, thus
achieving a better stabilization effect.
[0074] In the solid solution product of the present invention, an
additional stabilizing agent may be added, and other additives may
also be added as desired.
[0075] The present invention further provides another method for
preparing a solid solution. The method comprises dissolving
(6S)-5-methyl tetrahydrofolic acid or a salt thereof in water or an
aqueous solvent, adding a stabilizing agent to reverse the
impurities, and then crystallizing the stabilizing agent and
(6S)-5-methyl tetrahydrofolic acid or a salt thereof out,
filtering, and drying. Further, (6S)-5-methyl tetrahydrofolic acid
and the stabilizing agent are dispersed in water, sodium hydroxide
is added dropwise, and dissolved by stirring, then a corresponding
salt is added for crystallization, and the crystal is filtered out
and dried.
[0076] The present invention further provides a safe method of
using (6S)-5-methyl tetrahydrofolic acid or a salt thereof. The
method comprises a. making use of the composition comprising
(6S)-5-methyl tetrahydrofolic acid or a salt thereof, or the solid
solution; and b. adding a pharmaceutically acceptable reductive
substance as the stabilizing agent.
[0077] In a preferred safe method of using (6S)-5-methyl
tetrahydrofolic acid or a salt thereof, the stabilizing agent is
selected from vitamin C and an ester and salt thereof, isovitamin C
and an ester and salt thereof, dithiothreitol, reductive
glutathione, and zinc sulfate; preferably vitamin C and an ester
and salt thereof, and isovitamin C and an ester and salt thereof;
further preferably a sodium salt, a potassium salt, and a calcium
salt of vitamin C and isovitamin C, a magnesium salt of vitamin C
phosphate, and a sodium salt of vitamin C phosphate; and most
preferably vitamin C and isovitamin C.
[0078] In a preferred safe method of using (6S)-5-methyl
tetrahydrofolic acid or a salt thereof, the stabilizing agent is
combined with (6S)-5-methyl tetrahydrofolic acid by directly mixing
them in the form of solids, or by dissolving them in a solvent and
then crystallizing out together, or by preparing them into a
cocrystal or a solid solution, and preferably by preparing them
into a solid solution.
[0079] In the safe method of using (6S)-5-methyl tetrahydrofolic
acid or a salt thereof, the use of the stabilizing agent interrupts
the further progression of the oxidation of (6S)-5-methyl
tetrahydrofolic acid and the salt thereof. By means of this
solution, the negative effect of JK12A and other harmful
degradation products on human can be effectively controlled, and
the dosage can be accurately controlled. This safe method of using
is also referred to as METHOD JK. In the METHOD JK, the safety
characteristics of (6S)-5-methyl tetrahydrofolic acid can also be
ameliorated by using the two portions separately.
[0080] In a preferred safe method of using (6S)-5-methyl
tetrahydrofolic acid or a salt thereof, the weight ratio of the
stabilizing agent to (6S)-5-methyl tetrahydrofolic acid is
0.2-1000:1; and further preferably 1-10:1.
[0081] In the safe method of using (6S)-5-methyl tetrahydrofolic
acid or a salt thereof described in the present invention, vitamin
C and a salt thereof serve as the reducing agent, the salting-out
agent, and the stabilizing agent in a preferred embodiment. In
other words, vitamin C and the salt thereof are the reducing agent
for reversing the oxidized impurities, the salting-out agent used
in the purification process, and also the stabilizing agent for
(6S)-5-methyl tetrahydrofolic acid. Vitamin C and the salt thereof
remaining during production have no need to be removed dedicatedly.
In a subsequent process, a desired amount of vitamin C is added
when necessary. Also, similar effect can be achieved by using
reductive glutathione, zinc sulfate and a single isomer thereof,
dithiothreitol, isovitamin C, and so on. Logically, these
substances can be suitably used as the reducing agent, the
salting-out agent, and the stabilizing agent alone.
[0082] The safe method of using (6S)-5-methyl tetrahydrofolic acid
or a salt thereof is employed in the production of a preparation
comprising (6S)-5-methyl tetrahydrofolic acid alone or in
combination with other active ingredients. The preparation is
preferably in the form of common tablets, sustained release
tablets, controlled release tablets, effervescent tablets,
granules, common capsules, sustained release capsules, controlled
release capsules, dry suspensions, oral solutions, oral
suspensions, injections, and freeze-dried powder injections, and
further preferably in the form of common tablets, common capsules,
oral solutions, injections, and freeze-dried powder injections.
[0083] Use of the safe method of using (6S)-5-methyl
tetrahydrofolic acid or a salt thereof in the preparation of drugs
for treating or preventing diseases associated with deficiency of
5-methyl tetrahydrofolic acid or a salt thereof in mammals.
[0084] The present invention also provides use of the safe method
of using (6S)-5-methyl tetrahydrofolic acid or a salt thereof in
the preparation of drugs for treating or preventing diseases
associated with deficiency of 5-methyl tetrahydrofolic acid or a
salt thereof in mammals including, for example, subacute
encephalitis associated with dementia and vacuolar myelopathy,
cerebral folate deficiency, pathophysiology of vascular and
cardiovascular diseases such as premature obstructive arterial
disease, severe vascular disease in infancy and childhood,
progressive arterial stenosis, intermittent claudication, renal
vascular hypertension, ischemic cerebrovascular disease, premature
retinal artery and vein occlusion, cerebral occlusive arterial
disease, occlusive peripheral arterial disease, and premature death
due to thromboembolic disease and/or ischemic heart disease;
autoimmune diseases such as psoriasis, celiac disease, arthritis
and inflammatory conditions; magaloblastic anemia due to folate
deficiency, and intestinal absorption disorders; as antidotes for
folic acid antagonists (such as methotrexate, pyrimethamine or
trimethoprim, etc.); for preventing the severe toxic effects caused
by treatment with over or high dose of methotrexate; for reducing
the risk of abortion and/or giving birth to fetuses with neural
tube defects, cleft lip and/or cleft palate defects; for
maintaining normal homocysteine level and/or metabolism thereof;
changes in synthesis and functions of DNA and RNA, and in cell
synthesis; and depression.
[0085] The principle underlying the selection of the reducing
agent, the salting-out agent and the stabilizing agent of the
present invention includes primarily chemical safety such that the
product will not be degraded; and secondarily relative safety in
physiology. The substance that is fully validated in food and
medical science is preferred. It should be understood that the
amount of the reducing agent, the salting-out agent, and the
stabilizing agent is required to exceed a minimum amount, otherwise
it may cause the fact that the impurities cannot be sufficiently
reversed, the salting-out effect is poor and/or the stabilization
effect is poor. It should also be understood that in general, there
is no strict upper limit on the amount of the reducing agent and
the stabilizing agent.
[0086] The impurities contained in (6S)-5-methyl tetrahydrofolic
acid and a salt thereof according to the present invention include
JK12A and 5-methyl tetrahydropterioic acid having the following
structures:
##STR00006##
[0087] After oxidation, 5-methyl tetrahydropterioic acid is
converted into JK1303 having serious kidney toxicity with a high
yield, and thus need to be controlled strictly.
[0088] The present invention has the following beneficial effects.
The present invention provides high-purity (6S)-5-methyl
tetrahydrofolic acid or a salt thereof, the preparation method
therefor, the solid solution comprising high-purity (6S)-5-methyl
tetrahydrofolic acid or a salt thereof, the preparation method
therefor, and the safe method of using (6S)-5-methyl
tetrahydrofolic acid or a salt thereof. In all these embodiments,
the harmful chemicals possibly generated in a product of
(6S)-5-methyl tetrahydrofolic acid or a salt thereof are reversed
into the product itself by the use of a pharmaceutically acceptable
reducing agent, thus achieving the purpose of using (6S)-5-methyl
tetrahydrofolic acid or a salt thereof safely.
BRIEF DESCRIPTION OF THE DRAWINGS
[0089] FIG. 1 is a partially enlarged view of a large number of
vacuoles anatomically found after severe renal tubular necrosis of
the mice 14 days after administration of JK1303.
DETAILED DESCRIPTION
[0090] For better understanding of the technical solutions of the
present invention, the technical solutions of the present invention
will be further described below with reference to specific
embodiments; however the present invention is not limited
thereto.
[0091] The conditions used for detection by HPLC in the present
invention are as follows.
[0092] Chromatographic Conditions:
TABLE-US-00002 Chromatographic column C18-250 * 4.6 mm-5 .mu.m
Detection wavelength UV at 280 nm Flow rate 1.0 ml/min Injection
volume 20 .mu.l Column temperature 30.degree. C.
[0093] Formulation of Mobile Phases
[0094] Mobile phase A: 7.80 g of NaH.sub.2PO.sub.4.2H.sub.2O was
weighed, dissolved in 1000 ml of water, and adjusted to pH 6.5 with
a 32% NaOH solution.
[0095] Mobile phase B: 5.07 g of NaH.sub.2PO.sub.4.2H.sub.2O was
weighed, dissolved in 650 ml of water, added with 350 ml of
methanol, and adjusted to pH 8.0 with a 32% NaOH solution.
[0096] Gradient Program:
TABLE-US-00003 T (min) A % B % 0 100 0 14 45 55 17 0 100 22 0 100
31 100 0
[0097] Preparation of Sample Solution:
[0098] A suitable amount of a sample was dissolved in cold water at
2-8.degree. C. (which was prepared by adding sodium sulfite under
nitrogen atmosphere, distilling, collecting, and storing, and
cooled to 2-8.degree. C.) under nitrogen atmosphere, to formulate a
solution of about 0.5 mg/ml. The solution was prepared in situ when
used.
[0099] In some example of the present invention, ultrasonic waves
are used as an aid, which can obviously accelerate the reversion
and crystallization.
[0100] For ease of description, the data detected in the examples
is only with respect to 5-methyl tetrahydrofolic acid, salts
thereof, and related substances.
Example 1. Preparation of JK12A
[0101] Under nitrogen atmosphere, 10 g of (6S)-5-methyl
tetrahydrofolic acid was transferred to a reaction flask, and 80 g
of water was added, and adjusted to pH 7.3 with a 10% sodium
hydroxide solution with stirring. After the solid was completely
dissolved, 5 g of activated carbon was added, and reacted overnight
with stirring in open reactor. After the raw material was detected
to be reacted completely by HPLC, the reaction solution was
filtered, and the filtrate was adjusted to pH 4.0 with 50% acetic
acid. A crystal was formed, and filtered. The filter cake was
washed respectively with ethanol and acetone, and dried under
vacuum, to obtain 6.0 g of JK12A as a yellow solid (chemical purity
99.42%).
Example 2. Preparation of 5-Methyl Tetrahydropterioic Acid
[0102] 61 g of (6S)-5-methyl tetrahydrofolic acid was slowly added
to 600 ml of a 25% sodium hydroxide solution in water, heated to
reflux, and reacted overnight. After the raw material was reacted
completely, the reaction solution was cooled to room temperature,
and the impurities were filtered off. The filtrate was adjusted to
pH 9-10 with concentrated sulfuric acid, and the impurities were
precipitated out. The reaction solution was cooled to 10-15.degree.
C., and filtered. The filtrate was further adjusted to pH 2 with
concentrated sulfuric acid, and a solid was precipitated out,
stirred for 0.5 hr at 60.degree. C., and filtered while hot. The
solid was washed with water, and dried, to obtain 44 g of a white
solid (purity 96%).
Example 3. Preparation of JK1303
[0103] 5 g of (6S)-5-methyl tetrahydropterioic acid was fed to a
three-neck flask, 50 g of water was added, stirred fully, and
adjusted to pH 9.0 with a 10% sodium hydroxide solution. After the
solid was completely dissolved, 3 g of activated carbon was added,
the oxygen balloon was closed, and the stirring was continued
overnight. The reaction solution was filtered, and the filter cake
was washed with water. The filtrate was adjusted to pH 4 with a 50%
acetic acid solution in water, and the crystallization was
continued for 30 min with stirring. After filtering, the filter
cake was washed twice with water, slurried twice in 30 ml acetone,
and dried at 30.degree. C. under vacuum, to obtain 3.04 g of a
product (purity by HPLC: 94.3%).
Example 4. Reversion of JK12A
[0104] 10 g of water was added to 0.1 g of JK12A (purity by HPLC:
95.91%), and adjusted to pH 7.2 with 10% sodium hydroxide with
stirring. After the solid was completely dissolved, 0.8 g of
dithiothreitol was slowly added, and stirred for 1 hr. The reaction
solution was sampled and detected to contain 97.53% of 5-methyl
tetrahydrofolic acid and 0.72% of JK12A residue.
Example 5. Reversion of JK12A
[0105] 10 g of water was added to 0.1 g of JK12A (purity by HPLC:
95.91%), and adjusted to pH 7.2 with 10% sodium hydroxide with
stirring. After the solid was completely dissolved, 0.5 g of
cysteine was slowly added, and stirred for 1 hrs. The reaction
solution was sampled and detected to contain 96.64% of 5-methyl
tetrahydrofolic acid and 0.85% of JK12A residue.
Example 6. Reversion of JK12A
[0106] 0.1 g of JK12A (purity by HPLC: 74.04%) and 0.2 g of
mercaptoethanol were added to 5 ml of water, and adjusted to pH 7.5
with 30% sodium hydroxide with stirring. After the solid was
completely dissolved, stirring was further continued for 1 hr. Then
the reaction solution was sampled and detected to contain 60.92% of
5-methyl tetrahydrofolic acid and 0.59% of JK12A residue.
Example 7. Reversion of JK12A
[0107] 0.1 g of JK12A (purity by HPLC: 74.04%) and 0.2 g of sodium
mercaptoethane sulfonate were added to 5 ml of water, and adjusted
to pH 7.5 with 30% sodium hydroxide with stirring. After the solid
was completely dissolved, stirring was further continued for 1 hr.
Then the reaction solution was sampled and detected to contain
72.85% of 5-methyl tetrahydrofolic acid and 0.61% of JK12A
residue.
Example 8. Preparation and Conversion of Mixture Comprising
(6S)-Mefox and JK12A
[0108] Under nitrogen atmosphere, 1 g of (6S)-5-methyl
tetrahydrofolic acid was transferred to a reaction flask, and 8 g
of water was added, stirred, and adjusted to pH 7.3 with a 10%
sodium hydroxide solution. After the solid was completely
dissolved, 3 g of activated carbon was added, and reacted for 10
hrs with stirring in open reactor. The reaction solution was
detected by HPLC to contain 12.20% of 5-methyl tetrahydrofolic
acid; 83.51% of JK12A, and 0.57% of (6S)-Mefox), and filtered. The
filtrate was added with 10 g of vitamin C, and the pH was adjusted
to and maintained at 7.0 with caustic soda liquid. After stirring
for 3 hrs at 60.degree. C., the reaction solution was sampled and
detected to contain 96.49% of 5-methyl tetrahydrofolic acid; 0% of
JK12A; and 0% of (6S)-Mefox
Example 9. Reversion of JK1303
[0109] 10 g of water was added to 0.1 g of JK1303 (purity by HPLC:
96.7%), and adjusted to pH 7.0 with 10% sodium hydroxide with
stirring. After the solid was completely dissolved, 1 g of sodium
vitamin C was slowly added, and stirred for 1 hr. The reaction
solution was sampled and detected to contain 96.6% of 5-methyl
tetrahydropterioic acid and 0% of JK1303 residue.
Example 10. Composition Comprising (6S)-5-Methyl Tetrahydrofolic
Acid Calcium Salt
[0110] 50 ml of water and 50 g of vitamin C were neutralized to pH
7.0 with sodium hydroxide, and the solution became clear after the
solid was completely dissolved. 1 g of crude (6S)-5-methyl
tetrahydrofolic acid calcium salt was added batchwise, and
ultrasonicated at 65.degree. C. for 2 hrs. After filtering,
washing, and drying under vacuum, 0.72 g of a composition
comprising (6S)-5-methyl tetrahydrofolic acid calcium salt was
obtained.
TABLE-US-00004 Crude (6S)-5- methyl tetrahydrofolic acid calcium
Final Related substance salt/content product/content JK12A 0.49% --
5-methyl tetrahydrofolic acid 99.26% 99.9% 5-methyl
tetrahydropterioic acid 0.14% -- Other impurities 0.11% 0.01
[0111] The content is in percentages by weight, the same below.
Example 11. Composition Comprising (6S)-5-Methyl Tetrahydrofolic
Acid Calcium Salt
[0112] 15 g of (6S)-5-methyl tetrahydrofolic acid and 150 g of
vitamin C were added to 225 ml of water, and neutralized to pH 7.0
with a 30% sodium hydroxide solution in water. The solid was
dissolved, and the solution was stirred for 1 hr. Then 15 g of a
40% calcium chloride solution in water was added and ultrasonicated
at 70.degree. C. for crystallization. After 3 hrs, white particles
were crystallized out, filtered, and then washed with water. The
filter cake was dried under vacuum, to obtain 10.1 g of a solid
composition comprising (6S)-5-methyl tetrahydrofolic acid calcium
salt.
TABLE-US-00005 Crude (6S)-5-methyl Final Related substance
tetrahydrofolic acid/content product/content JK12A 0.22% 0.03%
(6S)-Mefox 0.02% 0.01% 5-methyl tetrahydrofolic 99.40% 99.95% acid
5-methyl tetrahydropterioic 0.03% -- acid Other impurities 0.33%
0.01%
Example 12. Composition Comprising (6S)-5-Methyl
Tetrahydrofolate
[0113] 1 g of (6S)-5-methyl tetrahydrofolic acid, 1 g of vitamin C,
and 0.5 g of NaCl were added to 10 ml of water, and neutralized to
pH 7.0 with a 30% sodium hydroxide solution in water. The solid was
dissolved, and the solution was stirred for 1 hr. Then 1 g of a 40%
calcium chloride solution in water was added and ultrasonicated at
70.degree. C. After 1 hr, a solid was crystallized out, filtered,
and then washed. The filter cake was dried under vacuum, to obtain
0.52 g of a solid composition comprising (6S)-5-methyl
tetrahydrofolic acid calcium salt.
TABLE-US-00006 Crude (6S)-5-methyl Final Related substance
tetrahydrofolic acid/content product/content JK12A 0.22% 0.07%
(6S)-Mefox 0.02% 0.01% 5-methyl tetrahydrofolic 99.40% 99.75% acid
5-methyl tetrahydropterioic 0.03% -- acid Other impurities 0.33%
0.15%
Example 13. Composition Comprising (6S)-5-Methyl Tetrahydrofolic
Acid Calcium Salt
[0114] 0.5 g of (6S)-5-methyl tetrahydrofolic acid, 10 mg of
vitamin C, and 0.1 g of sodium chloride were added to 10 ml of
water, and neutralized to pH 7.5 with a 30% sodium hydroxide
solution in water. The solution became clear after the solid was
completely dissolved. The reaction solution was stirred for 1 hr
under nitrogen atmosphere. 1 g of a 20% calcium chloride solution
in water was added, and ultrasonicated at 70.degree. C. for
crystallization. After 1.0 hr, white particles were crystallized
out, filtered, and then washed. The filter cake was dried under
vacuum, to obtain 0.38 g of a solid composition comprising
(6S)-5-methyl tetrahydrofolic acid calcium salt.
TABLE-US-00007 Crude (6S)-5-methyl Final Related substance
tetrahydrofolic acid/content product/content JK12A 0.22% 0.09%
(6S)-Mefox 0.02% 5-methyl tetrahydrofolic 99.40% 99.63% acid
5-methyl tetrahydropterioic 0.03% -- acid Other impurities 0.33%
0.27%
Example 14. Composition Comprising (6S)-5-Methyl Tetrahydrofolic
Acid Calcium Salt
[0115] 1 g of (6S)-5-methyl tetrahydrofolic acid and 3 g of
isovitamin C were added to 20 ml of water, and neutralized to pH
7.0 with a 30% sodium hydroxide solution in water. The solution
became clear after the solid was completely dissolved. The reaction
solution was stirred for 1 hr, and then 1 g of a 40% calcium
chloride solution in water was added, and ultrasonicated at
60.degree. C. After 1 hr, a solid were crystallized out, filtered,
and then washed. The filter cake was dried under vacuum, to obtain
0.80 g of a composition comprising (6S)-5-methyl tetrahydrofolic
acid calcium salt.
TABLE-US-00008 Crude (6S)-5-methyl Final Related substance
tetrahydrofolic acid/content product/content JK12A 0.22% --
(6S)-Mefox 0.02% 0.05% 5-methyl tetrahydrofolic 99.40% 99.75% acid
5-methyl tetrahydropterioic 0.03% -- acid Other impurities 0.33%
0.18%
Example 15. Composition Comprising (6S)-5-Methyl Tetrahydrofolic
Acid
[0116] 0.5 g of the composition comprising calcium (6S)-5-methyl
tetrahydrofolate prepared in Example 11 was heated to 40.degree.
C., and adjusted to pH 4.0 with a hydrochloric acid solution. The
system was stirred for 1 hr for crystallization while the pH was
maintained unchanged. After cooling to room temperature, filtering,
washing, and drying at 25.degree. C. under vacuum, 0.23 g of a
composition comprising (6S)-5-methyl tetrahydrofolic acid was
obtained, which contains 99.80% of (6S)-5-methyl tetrahydrofolic
acid, 0% of JK12A, 0.07% of (6S)-Mefox, and 0% of 5-methyl
tetrahydropterioic acid, as detected by HPLC.
TABLE-US-00009 Composition of (6S)-5- methyl tetrahydrofolic acid
Final Related substance calcium salt/content product/content JK12A
0.03% -- (6S)-Mefox 0.01% -- 5-methyl tetrahydrofolic 99.95% 99.80%
acid 5-methyl tetrahydropterioic -- -- acid Other impurities 0.01%
0.20%
Example 16. Composition Comprising 6R,S-5-Methyl Tetrahydrofolic
Acid Calcium Salt
[0117] 1 g of 6R,S-5-methyl tetrahydrofolic acid and 10 g of
vitamin C were added to 15 ml of water, and neutralized to pH 7.0
with a 30% sodium hydroxide solution in water. The solution became
clear after the solid was completely dissolved. 1 g of a 40%
calcium chloride solution in water was added, and ultrasonicated at
70.degree. C. for crystallization. After filtering, washing with
water, and drying under vacuum, 0.54 g of a composition comprising
6R,S-5-methyl tetrahydrofolate was obtained.
TABLE-US-00010 Crude 6R, S-5-methyl Final Related substance
tetrahydrofolate/content product/content JK12A 0.21% -- (6S)-Mefox
-- -- 5-methyl tetrahydrofolic acid 99.15% 99.59% 5-methyl
tetrahydropterioic 0.28% 0.08% acid Other impurities 0.36%
0.33%
Example 17. Composition Comprising Sodium 6R,S-5-Methyl
Tetrahydrofolic Acid
[0118] 1 g of 6R,S-5-methyl tetrahydrofolic acid, and 5 g of
vitamin C were added to 20 ml of water, and neutralized to pH 7.0
with a 30% sodium hydroxide solution in water. The solution became
clear after the solid was completely dissolved. The reaction was
continued for 2 hrs at 60.degree. C. with oxygen being isolated.
Then, the reaction solution was added dropwise to 200 ml of
ethanol, to crystallize a sodium salt out, which was filtered,
washed, and dried under vacuum, to obtain 0.52 g of a composition
comprising sodium 6R,S-5-methyl tetrahydrofolate.
TABLE-US-00011 Crude 6R, S-5-methyl Final Related substance
tetrahydrofolic acid/content product/content JK12A 0.21% --
(6S)-Mefox -- -- 5-methyl tetrahydrofolic 99.15% 99.64% acid
5-methyl tetrahydropterioic 0.28% 0.12% acid Other impurities 0.36%
0.34%
Example 18. Composition Comprising of (6S)-5-Methyl Tetrahydrofolic
Acid Arginine Salt
[0119] 5 g of vitamin C was added to 5 ml of water, and neutralized
to pH 7.0 with sodium hydroxide. The solution became clear after
the solid was completely dissolved. 0.5 g of crude (6S)-5-methyl
tetrahydrofolic acid arginine salt was added, dissolved completely,
and ultrasonically stirred for 2 hrs at 60.degree. C. The reaction
solution was added dropwise to 50 ml of ethanol, and stirred to
crystallize a solid out. The solid was filtered out and detected.
0.26 g of a composition comprising (6S)-5-methyl tetrahydrofolic
acid arginine salt was obtained.
TABLE-US-00012 Crude 5-methyl tetrahydrofolic acid Final Related
substance arginine salt/content product/content JK12A 0.76% --
(6S)-Mefox 1.30% 0.27% 5-methyl tetrahydrofolic acid 97.69% 99.70%
5-methyl tetrahydropterioic acid 0.05% -- Other impurities 0.20%
0.03%
Example 19. Composition Comprising (6S)-5-Methyl Tetrahydrofolic
Acid D-Glucosamine Salt
[0120] 5 g of vitamin C was added to 10 ml of water, and
neutralized to pH 7.0 with sodium hydroxide. The solution became
clear after the solid was completely dissolved. 2.0 g of crude
(6S)-5-methyl tetrahydrofolic acid D-glucosamine salt was added,
dissolved completely, and ultrasonically stirred for 2 hrs at
60.degree. C. Ethanol was slowly added dropwise, and a solid was
crystallized out, which was filtered and recrystallized in ethanol,
to obtain a composition comprising (6S)-5-methyl tetrahydrofolic
acid D-glucosamine salt.
TABLE-US-00013 Crude 5-methyl tetrahydrofolic acid Final Related
substance D-glucosamine salt/content product/content JK12A 0.56% --
(6S)-Mefox 0.70% 0.14 5-methyl tetrahydrofolic 98.47% 99.81% acid
D-glucosamine salt 5-methyl tetrahydropterioic 0.05% -- acid Other
impurities 0.22% 0.05%
Example 20. Solid Solution Comprising (6S)-5-Methyl Tetrahydrofolic
Acid Calcium Salt and Vitamin C
[0121] 1 g of (6S)-5-methyl tetrahydrofolic acid and 2.0 g of
vitamin C were added to 20 ml of water, and neutralized to pH 7.5
with a 30% sodium hydroxide solution in water. The solid was
dissolved, and the solution was stirred for 1 hr. Then 1 g of a 40%
calcium chloride solution in water was added and ultrasonicated at
70.degree. C. for crystallization. After 1.0 hr, white particles
were crystallized out, filtered, dried, and detected by HPLC to
comprise 94.89% of (6S)-5-methyl tetrahydrofolic acid calcium salt,
and 5.11% of vitamin C.
Example 21: Solid Solution Comprising (6S)-5-Methyl Tetrahydrofolic
Acid Calcium Salt and Isovitamin C
[0122] 0.1 g of (6S)-5-methyl tetrahydrofolic acid and 0.3 g of
isovitamin C were added to 1.5 ml of water, and neutralized to pH
7.5 with a 30% sodium hydroxide solution in water. The solid was
dissolved, and the solution was stirred for 1 hr. Then 1 g of a 40%
calcium chloride solution in water was added and ultrasonicated at
90.degree. C. for crystallization. After 1.0 hr, white particles
were crystallized out, which were filtered, dried, and detected by
HPLC to comprise 29.29% of (6S)-5-methyl tetrahydrofolic acid
calcium salt, and 70.66% of isovitamin C.
Example 22. Solid Solution Comprising (6S)-5-Methyl Tetrahydrofolic
Acid Calcium Salt and Zinc Sulfate
[0123] 0.5 g of (6S)-5-methyl tetrahydrofolic acid and 1.0 g of
zinc sulfate were added to 10 ml of water, and neutralized to pH
7.5 with a 30% sodium hydroxide solution in water. The solid was
dissolved, and the solution was stirred for 1 hr. Then 1 g of a 20%
calcium chloride solution in water was added and ultrasonicated at
70.degree. C. for crystallization. After 1.0 hr, a solid solution
was obtained, which was filtered, dried, and detected by HPLC (with
an ELSD detector) to comprise 27.69% of (6S)-5-methyl
tetrahydrofolic acid calcium salt, and 72.26% of zinc sulfate.
Example 23. Solid Solution Comprising 5-Methyl Tetrahydrofolic Acid
Calcium Salt and Reductive Glutathione
[0124] 0.5 g of (6S)-5-methyl tetrahydrofolic acid and 1.5 g of
reductive glutathione were added to 10 ml of water, and neutralized
to pH 7.5 with a 30% sodium hydroxide solution in water. The solid
was dissolved, and the solution was stirred for 1 hr. Then 1 g of a
20% calcium chloride solution in water was added and ultrasonicated
at 70.degree. C. for crystallization. After 1.0 hr, white particles
were crystallized out, which were filtered, dried, and detected by
HPLC (with an ELSD detector) to comprise 37.62% of (6S)-5-methyl
tetrahydrofolic acid calcium salt, and 62.33% of reductive
glutathione.
Example 24. Solid Solution Comprising (6S)-5-Methyl Tetrahydrofolic
Acid and Vitamin C
[0125] 0.5 g of (6S)-5-methyl tetrahydrofolic acid and 3.0 g of
vitamin C were added to 10 ml of water, and neutralized to pH 7.5
with a 30% sodium hydroxide solution in water. The solid was
dissolved, and the solution was stirred for 1 hr. A suitable amount
of 6N hydrochloric acid solution in water was added dropwise, to
adjust the pH to 4.0, and stirred 50.degree. C. for
crystallization. After 1.0 hr, white particles were crystallized
out, which were filtered, dried, and detected by HPLC to comprise
26.80% of (6S)-5-methyl tetrahydrofolic acid and 73.17% of vitamin
C.
Example 25. Solid Solution Comprising (6S)-5-Methyl Tetrahydrofolic
Acid Zinc Salt and Vitamin C
[0126] 0.5 g of (6S)-5-methyl tetrahydrofolic acid and 1.5 g of
vitamin C were added to 10 ml of water, and neutralized to pH 7.5
with a 20% sodium hydroxide solution in water. The solution became
clear after the solids were completely dissolved. 0.8 g of a 50%
zinc chloride solution in water was added, crystallized for 2 hrs,
and filtered under nitrogen atmosphere. The filter cake was dried
under vacuum and detected by HPLC to comprise 88.7% of
(6S)-5-methyl tetrahydrofolic acid zinc salt, and 11.0% of vitamin
C.
Example 26: Solid Solution of (6S)-5-Methyl Tetrahydrofolic Acid
Calcium Salt, and Magnesium Vitamin C Phosphate
[0127] 0.5 g of (6S)-5-methyl tetrahydrofolic acid, and 2.0 g of
magnesium vitamin C phosphate were added to 10 ml of water, and
neutralized to pH 7.5 with a 20% sodium hydroxide solution in
water. The solution became clear after the solids were completely
dissolved. 0.6 g of a 50% calcium chloride solution in water was
added, and then reacted ultrasonically at 70.degree. C. After 1.5
h, white particles were crystallized out, which were filtered under
nitrogen atmosphere, dried and detected by HPLC to comprise 62.4%
of (6S)-5-methyl tetrahydrofolic acid calcium salt, and 37.4% of
vitamin C.
Example 27. Stability of Composition Comprising (6S)-5-Methyl
Tetrahydrofolic Acid Calcium Salt Prepared Through METHOD JK and
Vitamin C
[0128] 1 g of the composition of (6S)-5-methyl tetrahydrofolic acid
calcium salt obtained in Example 11 was fully mixed by grinding
with vitamin C in various proportions by weight, placed in an
incubator at 25.degree. C. and 40% humidity, and detected for
(6S)-5-methyl tetrahydrofolic acid calcium salt and JK12A
periodically.
TABLE-US-00014 Amount of VC 0 20% 100% (6S)-5-methyl (6S)-5-methyl
(6S)-5-methyl tetrahydrofolic JK1 tetrahydrofolic JK1
tetrahydrofolic JK1 Days acid calcium salt 2A acid calcium salt 2A
acid calcium salt 2A 0 99.95% 0.03% 99.95% 0.03% 99.95% 0.03% 7
99.90% 0.06% 99.94% 0.04% 99.94% 0.03% 15 99.85% 0.12% 99.89% 0.08%
99.90% 0.05% 30 99.63% 0.25% 99.71% 0.17% 99.90% 0.05% 60 99.40%
0.46% 99.50% 0.22% 99.86% 0.10% Amount of VC 500% 1000% 10000%
(6S)-5-methyl (6S)-5-methyl (6S)-5-methyl tetrahydrofolic JK1
tetrahydrofolic JK1 tetrahydrofolic JK1 Days acid calcium salt 2A
acid calcium salt 2A acid calcium salt 2A 0 99.95% 0.03% 99.95%
0.03% 99.95% 0.03% 7 100.00% -- 100.00% -- 100.00% -- 15 100.00% --
100.00% -- 100.00% -- 30 99.98% -- 100.00% -- 100.00% -- 60 99.96%
-- 100.00% -- 100.00% --
* * * * *